Functional analysis of the extended N-terminus for the Drosophila Raf protein and initial characterization of the Arl1 gene.

摘要

The Raf serine/threonine kinases play a key role(s) in receptor tyrosine kinase (RTK) signaling. However, the mechanisms that modulate Raf activity are complicated and remain elusive. Clues to Raf regulation were derived from the identification of conserved regions/motifs/sites. Drosophila Raf (DRaf) has an extended N-terminus. We show this N-terminal segment contains a novel region (CRN) that is conserved in BRaf proteins of vertebrates. The extended N-terminus can contribute to Torso RTK signaling during embryogenesis in both loss and gain-of-function genetic backgrounds. Furthermore, stronger interactions between DRaf's RBD (Ras Binding Domain) and the small GTPase Ras1, as well as Rap1, were observed in vitro when CRN and RBD sequences were linked. Together, these studies suggest that the N-terminal segment may assist in the association of DRaf with its activators (Ras1 and Rap1) and play a positive regulatory role(s) in DRaf activation in vivo through CRN-mediated mechanisms.;We also tried to characterize the roles of DRaf's conserved CR2 domain. In our yeast 2-hybrid screen using CR2 as the bait, a putative DRaf binding partner, small GTPase Arf-like1 (Arl1) protein, was identified. Using genetics approaches we have made an effort to understand the roles of Arl1 in Drosophila. Our studies suggest that Arl1 may function in membrane traffic.